Genome Editing of Veterinary Relevant Mycoplasmas Using a CRISPR-Cas Base Editor System

ABSTRACT Mycoplasmas are minimal bacteria that infect humans, wildlife, and most economically relevant livestock species. Mycoplasma infections cause a large range of chronic inflammatory diseases, eventually leading to death in some animals. Due to the lack of efficient recombination and genome engineering tools for most species, the production of mutant strains for the identification of virulence factors and the development of improved vaccine strains is limited. Here, we demonstrate the adaptation of an efficient Cas9-Base Editor system to introduce targeted mutations into three major pathogenic species that span the phylogenetic diversity of these bacteria: the avian pathogen Mycoplasma gallisepticum and the two most important bovine mycoplasmas, Mycoplasma bovis and Mycoplasma mycoides subsp. mycoides. As a proof of concept, we successfully used an inducible SpdCas9-pmcDA1 cytosine deaminase system to disrupt several major virulence factors in these pathogens. Various induction times and inducer concentrations were evaluated to optimize editing efficiency. The optimized system was powerful enough to disrupt 54 of 55 insertion sequence transposases in a single experiment. Whole-genome sequencing of the edited strains showed that off-target mutations were limited, suggesting that most variations detected in the edited genomes are Cas9-independent. This effective, rapid, and easy-to-use genetic tool opens a new avenue for the study of these important animal pathogens and likely the entire class Mollicutes. IMPORTANCE Mycoplasmas are minimal pathogenic bacteria that infect a wide range of hosts, including humans, livestock, and wild animals. Major pathogenic species cause acute to chronic infections involving still poorly characterized virulence factors. The lack of precise genome editing tools has hampered functional studies of many species, leaving multiple questions about the molecular basis of their pathogenicity unanswered. Here, we demonstrate the adaptation of a CRISPR-derived base editor for three major pathogenic species: Mycoplasma gallisepticum, Mycoplasma bovis, and Mycoplasma mycoides subsp. mycoides. Several virulence factors were successfully targeted, and we were able to edit up to 54 target sites in a single step. The availability of this efficient and easy-to-use genetic tool will greatly facilitate functional studies of these economically important bacteria.

unwanted BseRI restriction sites were performed before synthesis of the DNA fragments by Twist Bioscience.
Plasmid construction. The Mini-Tn4001tet 5 and the synthesized fragments provided by Twist Biosciences were used as PCR templates. The Q5 High-Fidelity DNA Polymerase kit (NEB, M0491) was used for all PCR reactions. Primers A1/A2 (see Supplementary Table S1) were used to amplify the plasmid backbone encoding the transposon and the tetracycline repressor; primers A3/A4 for the synthetized fragment encoding the sgRNA and the first 665 amino-acids of the hybrid protein dCas9-deaminase-UGI; and primers A5/A6 for the synthesized fragment encoding the last 1067 amino-acids of the dCas9-deaminase-UGI hybrid protein and the puromycin resistance marker 6 .
PCR products were incubated with the DpnI restriction enzyme (NEB, R0176S) following the manufacturer's recommendations. Both products were purified using the GFX PCR DNA or Gel Band Purification Kit (Cytiva). The NEBuilder HiFi DNA Assembly Cloning Kit (NEB, E5520S) was used to assemble the DNA fragments and create the circular plasmids pTi4.0_rAPOBEC1_ SpdCas9 and pTi4.0_ SpdCas9_pmcDA1. E. coli NEB 5α (NEB, C2987H) was transformed with 2 µL of the assembled constructs. Transformants were screened after DNA extraction with the NucleoSpin Plasmid kit (Macherey-nagel, 740588.50) and enzymatic digestion. Sanger sequencing of the targeted locus was performed (Genewiz) for final verification.
For the plasmid pMT85_spCas9-pmcDA1, the pMT85-2Res-Genta backbone 7 was used instead of the Mini-Tn4001 transposon backbone. Primers G38/G39 were used to amplify a fragment containing the sgRNA, deaminase complex, and tetracycline repressor. Primers G36/G37 were used to amplify the pMT85-2Res-Genta backbone. Cloning was performed using the same procedure as for pTi4.0 (see above).
For the plasmid pMYCO1_spCas9-pmcDA1, the pMYCO1 backbone (oriC plasmid 8 ) was used instead of the transposon backbone. Primers D27/D28 were used to amplify a fragment containing the sgRNA, deaminase complex, tetracycline repressor, and puromycin resistance marker, and primers D29/D30 were used to amplify the pMYCO1 backbone. Cloning was performed using the same procedure as for pTi4.0 (see above).
For all CBE constructs, 20-nucleotides targets were added using the following process. The targets were designed as oligonucleotides R and F. Ten microliters of each primer (100 µM) were mixed with 2 µL Adv2 polymerase buffer (TAKARA, 639232) and heated at 95°C for 5 min, followed by slowl cooling (-0.1°C/sec) to room temperature to anneal the complementary oligonucleotides.
Plasmids were digested with BseRI (NEB, R0581S) for 2 h at 37°C. After purification, target sequences and linearized plasmids were ligated using T4 DNA ligase (Promega) overnight at 4°C. NEB 5α competent E. coli (NEB, C2987H) was transformed with 2 µL of the ligation mix. Colonies were screened by digestion of the plasmids with BseRI and KpnI restriction enzymes and the target sequences verified by Sanger sequencing (Genewiz).

Induction of CBE.
For each mycoplasma species, transformants were picked and grown in selective liquid media for three passages (one passage is equivalent to a 1:100 dilution). At passage 3, fresh aTC (Abcam, ab145350) (in EtOH 50%) was added to the culture at early logarithmic growth phase (~10 h for M. bovis and Mmm, ~24 h for M. gallisepticum) and the cells were grown until the stationary phase was reached (12 h after induction for M. bovis, 18 h for Mmm, 24 h for M. gallisepticum). An aTC concentration of 0.5 µg.mL -1 was used for the three mycoplasma species. After induction, cells were plated on selective solid medium. Isolated clones were obtained after incubation at 37°C for 3 to 10 days. Alternatively, induction was performed immediately after transformation. In this case, after the 2 h incubation step at 37°C (recovery of the cells), antibiotics (puromycin or gentamicin) were added to the media and the cultures incubated for 2 h. Then, the base-editor system was induced using fresh aTC (0.5 µg.mL -1 ) for 12 h to 15 h (overnight). Induced cultures were plated on selective media and incubated at 37°C with 5% CO2.
PCR screening of transformants. PCR screening was performed on all transformants before induction, after induction, or on isolated clones using the Advantage HF 2 PCR Kit (Takara, 639123). A list of all primers used to screen sites of deamination is available in Table S1.
EditR analysis. EdiTR 1.0.10 software (https://moriaritylab.shinyapps.io/editr_v10/) was used to analyze and quantify base editing at each position of the 20-nucleotide target 9 . The analysis was performed using the ".ab" files generated by Sanger sequencing.
Whole genome sequencing of mycoplasmas. Genomic DNA of M. gallisepticum was extracted from a 10 mL culture using the Qiagen Genomic-Tips 100/G kit. Genome sequencing was performed by the Genome Transcriptome Facility of Bordeaux. Long reads were produced using a GridION device (Oxford Nanopore) and short reads a MiSeq device (Illumina). For Mmm_glpO mutant cl18_4, ONT sequencing generated 24,400 reads (mean read length: 29,377 bp) and Illumina 1,044,350 read pairs. Analyses were performed using Galaxy (https://usegalaxy.eu/).  Table S3. Genome assembly was performed using the following steps: ONT reads were filtered using Filter FASTQ (V 1.1.5, Minimum size 45,000 bp), assembled using Flye Assembly (V 2.6), and polished using four rounds of Pilon (1.20.1) combined with Illumina short reads. The assembled genome was compared to the Mmm T1/44 (CP014346.1) reference genome using MAUVE software 10  All SNPs found in sequenced clones were classified to identify their potential origin. A summary can be found in Table S3. For sgRNA-mediated SNPs, a Blast of the target sequence against the DNA region around the SNP was performed. Two conditions were established to consider a single nucleotide variation (SNV) as an sgRNA-dependent off-target mutation: (1) the presence of the PAM sequence and (2) at least 70% sequence similarity for the first 12 nucleotides (considered as the Seed-sequence). For other mutations, C to T or G to A SNPs were considered to result from spurious deamination and the nucleotide before was observed to be a TC motif, which are preferred by the deaminase protein.
Phenotypic assay for M. bovis : nuclease activity. Nuclease activity assays on WT M. bovis PG45 and the mutant Mbov_mnuA were performed as previously described in Sharma et al. 2015 11 with several modifications. Briefly, for each strain, 2 mL of culture was prepared in SP4 medium.
At late log phase, cultures were divided in half and centrifuged for 10 min at 7,000 x g at 10°C.
One 1-mL sample was mixed with 500 µL nuclease buffer (25 mM Tris-HCl, pH 8.8, 10 mM CaCl2, 10 mM MgCl2) and the other 1-mL sample was mixed with 100 µL nuclease buffer to concentrate the mycoplasma cells (2 and 10 times, respectively). Then, 2 µg circular plasmid DNA (pMT85_SpCas9_pmcDA1) or 500 ng double stranded linear DNA (PCR fragment) was incubated with 50 µL of each sample at 37°C for 5 or 60 min. At each time point, 10-µL aliquots were removed and the reaction was stopped by the addition of EDTA to a final concentration of 20 mM. Aliquots were mixed with 6X loading buffer (Promega) and immediately loaded onto 1% agarose gels made in 1X TAE buffer.
Phenotypic assay for Mmm: H2O2 production. Mmm T1/44 WT and Mmm_glpO mutants were grown until reaching a pH of 7. A 20-µL drop of each culture was then plated on PPLO media and the plates incubated at 37°C for 48 h. GlpO activity was evaluated using a qualitative "on-theplate" H2O2 test that allows the detection of H2O2 production in response to the addition of a glycerol-containing reaction mix 12  ), KCl (1.5 mg.mL -1 ), NaHCO3 (0.625 mg.mL -1 ), NaCl (56.25 mg.mL -1 )]) was carefully spread onto bacterial lawns and the colorimetric reaction observed after 30 min or 2 h of incubation at 37°C. Mycoplasma colonies producing H2O2 adopt a red-brown color.